Liposome dynamin effect

Fig. 3. Dynamin s liposome-stimulated GTPase activity varies with liposome composition. (A) Dynamin s GTPase activity is greater when assayed in the presence of liposomes composed exclusively of DOPS, compared to liposomes prepared from DOPC PI4,5P2 (90 10 mol%). Liposomes composed of a higher mol% PI4,5P2 (see Fig. 2) are significantly more effective templates. (B) Negative-stain electron micrographs of dynamin-generated lipid tubules formed on DOPS and DOPC PI4,5P2 liposomes are indistinguishable, despite the observed differences in rates of GTP hydrolysis. Scale bar = 50 nm.

Fig. 2. GTPase activity of dynamin with amphiphysin. Dynamin GTPase activity was assayed in the presence of large unilamellar liposome (A) or small liposome (B). Note that the different effect of amphiphysin on dynamin GTPase activity was observed by the size of liposomes. (Reproduced with permission from Y. Yoshida et al [2004]. EMBO J. 23, 3483-3491.)...

Thus, the effect of amphiphysin 1 on dynamin GTPase activity is strongly affected by the size of the liposomes. Absolute GTPase activity of dynamin was much higher with small liposomes than with large liposomes (Fig. 2B), probably because the surface area of liposomes has been increased by sonication. 

Fig. 2. Examples of the dynamin I colorimetric GTPase assay. (A) L-a-phosphatidyl-L-serine (PS) liposome concentration curve. Stimulation of dynamin I GTPase activity was measured as a function of PS concentration using the colorimetric assay. The curve reaches a plateau at about 30 /xg/ml PS in this experiment, but varies between PS preparations. The curve is representative of the level of optimal dynamin activity in the absorbance range 0.4-0.6 OD units. (B) Concentration-dependent effect of compound A on dynamin I GTPase activity stimulated by 40 /xg/ml PS. The effect of the drug alone at increasing concentrations is shown in the open bars. The stimulation by PS + Compound A is shown in the hatched bars. Compound A reduces PS-stimulated dynamin I GTPase activity (hatched bars), until at high concentrations there appears to be stimulation. The solid bars show a subtraction of the other values, which, after eliminating the effect of background phosphate, reveals the full inhibitory curve of compound A.

Fig. 1. studying dynamin, amphiphysin, and epsin mediated liposome tabulation. (A) An exploded view of the lipid extrusion devise used for the formation of hposomes. (B) Liposomes formed from Folch brain Upid extract. (C) The effect of dynamin on Folch brain hpid hposomes. (D) Tubules formed by incubating hposomes with Drosophila amphiphysin. (E) Tubules formed by incubating the ENTH domain of epsin with liposomes. The scale bars are 100 nm. 

Different lipid molecules associate together to form membrane structures with very different overall curvatures. When liposomes are made in the presence of sufficient concentrations of nonhydroxylated fatty-acid galactoceramides (NFA-GalCer) they form nanotubes (Wilson-Kubalek et al, 1998). We have used these preformed lipid nanotubes to examine the effect of GTP hydrolysis on the conformation of a dynamin helix (Marks et al, 2001 Stowell et al, 1999). Indeed, in the absence of GTP hydrolysis, purified dynamin aggregates to form helices and, as seen above, can tubulate liposomes. Also, dynamin interacts with lipid nanotubes to form ordered arrays with a diameter similar to the constricted necks of clathrin-coats pits observed in vivo (Stowell et al, 1999 Takei et al, 1995), suggesting that the nanotube acts as a good mimic of the neck of a vesicle. 

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